The fragmentation and tagging of DNA (e.g., genomic DNA) is an important step in DNA sample preparation for high-throughput sequencing, also referred to as next generation sequencing (NGS). Earlier sample preparation methods, such as DNA fragmentation using DNAse I, are very unreliable and often result in DNA fragmentation that is either insufficient or too extensive. In either case, the yield of DNA fragments of useful size (about 200-800 base pairs (bp)) is low. DNA shearing using sonicators, for example E220 and E220x instruments from Covaris (Woburn, Mass.), provide an alternative. However, such instruments are very expensive (over $100,000 in 2012 prices) and overall DNA shearing is a laborious and multi-stage process. It involves DNA fragmentation, fragments ends repair, first fragments purification, poly-A tailing, adapter ligation, second fragments purification, PCR amplification, and third fragments purification.
A number of steps in this process can be eliminated/combined using oligonucleotide-transposase complexes, such as the NEXTERA™ DNA sample prep kit from IIlumina (San Diego, Calif.). Oligonucleotide-transposase complexes can effect both controlled DNA fragmentation and attachment of adapters in a single reaction, which takes only a few minutes. Examples of such transposase complexes include those that contain a dimer of modified Tn5 transposase and a pair of Tn5-binding double-stranded DNA (dsDNA) oligonucleotides containing a 19 bp transposase-binding sequence, or inverted repeat sequence (IR). In the NEXTERA™ system mentioned above, an engineered, non-native 19 bp transposase binding sequence is used, which provides more efficient DNA fragmentation and tagging than the native Tn5 IR sequence. This binding sequence is referred to as “mosaic”.
Unlike DNAase, a single molecule of which can generate numerous breaks in a target DNA, the transposase complex is believed to create only one DNA cleavage per complex. Therefore, unlike with DNAse I, the degree of DNA fragmentation is easily controlled during transposase fragmentation by controlling the ratio of transposase complex to target DNA in the reaction mixture. Furthermore, specific nucleotide tags combined with the mosaic sequence can be attached in this transposase-mediated DNA fragmentation process, which is useful for DNA amplification in PCR and attaching the tagged DNA fragments to sequencing chips.
Given the ever increasing capabilities of NGS technologies to sequence and deconvolute thousands of different barcoded DNA samples mixed in the same sequencing reaction, there is a need in the art to provide improved methods for transposase-mediated barcoding and fragmentation of multiple individual DNA samples. The present disclosure provides methods and compositions that find use in this, and other, applications.